Subpopulations of Spore-Like Cells and Uses Thereof

ABSTRACT

Subpopulations of spore-like cells expressing specific cell surface and gene expression markers are provided. In one embodiment, the cells express at least one cell surface or gene expression marker selected from the group consisting of Oct4, nanog, Zfp296, cripto, Gdf3, UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1, CD29, CD34, CD90, B1 integrin, cKit, SP-C, CC10, SF1, DAX1, and SCG10. Also provided are methods for purifying a subpopulation of spore-like cells of interest from a population of spore-like cells, and methods for inducing differentiation of the isolated spore-like cells into cells of endodermal, mesodermal or ectodermal origin. The spore-like cells can be used to generate cells originating from all three germ layers and can be used to treat a patient who has a deficiency of functional cells in any of a wide variety of tissues, including the retina, intestine, bladder, kidney, liver, lung, nervous system, or endocrine system.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application Ser. No. 61/260,607filed Nov. 12, 2009.

FIELD OF THE INVENTION

The invention relates to the identification, isolation and uses ofsubpopulations of spore-like cells.

BACKGROUND OF THE INVENTION

The use of pluripotent cells has gained interest in medical research,particularly in the area of providing reagents for treating tissuedamage either as a result of genetic defects, injuries, and/or diseaseprocesses. Ideally, cells that are capable of differentiating into theaffected cell types could be transplanted into a subject in needthereof, where they would interact with the organ microenvironment andsupply the necessary cell types to repair the injury. Embryonic stem(ES) cells are pluripotent cells derived from blastocysts that can bepropagated indefinitely undifferentiated in vitro, can differentiate toall cell lineages in vivo, and can be induced to differentiate into mostcell types in vitro (Martin, Proc Natl Acad Sci U S A., 78:7634-7638(1981)). Although ES cells have been isolated from humans, their use inresearch as well as therapeutics is encumbered by ethical considerations(Frankel, Science. 287:1397 (2000)

There have been increasing efforts to isolate stem cells fromnon-embryonic tissues, including hematopoietic (U.S. Pat. No.5,750,397), neural, Gage, Science, 287:1433-1438 (2000),gastrointestinal, Potten, Philos Trans R Soc Lond B Biol Sci.353:821-830 (1998), epidermal, (Watt, Philos Trans R Soc Lond B BiolSci., 353:831-837 (1998), and mesenchymal stem cells (MSCs) (U.S. Pat.No. 5,736,396). Another population of cells, multipotent adultprogenitor cells (MAPCs), has also been purified from bone marrow (Reyeset al., Blood, 98(9):2615-2625 (2001); Reyes & Vetfaillie, Ann NY AcadSci., 938:231-235 (2001)). These cells are capable of expansion in vitrofor more than 100 population doublings without telomere shortening orthe development of karyotypic abnormalities. MAPCs have also been shownto be able to differentiate under defined culture conditions intovarious mesenchymal cell types (e.g., osteoblasts, chondroblasts,adipocytes, and skeletal myoblasts), endothelium, neuroectoderm cells,and more recently, into hepatocytes (Schwartz et al., Clin Invest,109:1291-1302 (2000)).

A challenge to the use of ES cells or other pluripotent cells forregenerative therapy in a subject is to control the growth anddifferentiation of the cells into the particular cell type required fortreatment of a subject. As disclosed in Schuldiner et al., Proc NatlAcad Sci USA, 97:11307-11312 (2000), none of the eight growth factorsused therein directed differentiation exclusively to one cell type.Thus, there continues to be a need for new approaches to generatepopulations of transplantable multi- and pluripotent cells suitable fora variety of applications, including treating injury and/or disease ofvarious organs and/or tissues. Furthermore, sources of the multipotentor pluripotent cells are limited in that the cells must be harvestedfrom living tissue.

U.S. Published Application Nos. 2004/0057942 and 2004/0033598 byVacanti, et al., disclose small primitive cells which have anexceptionally high tolerance for oxygen deprivation. These cells, called“spore-like” cells, have been demonstrated to tolerate essentiallycomplete oxygen deprivation for at least 24 hours (cells were viabledespite oxygen deprivation for either four or 24 hours). Spore-likecells have a greater capacity to proliferate than terminallydifferentiated cells isolated from specialized tissues. Proliferativecapacity is an important attribute because tissue engineering, celltherapies, and gene-based therapies are often hampered by physicians'inability to obtain sufficient numbers of cells to administer to apatient.

It is therefore an object of the present invention to providesubpopulations of spore-like cells expressing specific cell surface orgene expression markers.

It is also an object of this invention to provide a subpopulation ofpluripotent spore-like cells, and a method for isolating a subpopulationof pluripotent spore-like cells.

SUMMARY OF THE INVENTION

Sub-populations of spore-like cells expressing specific cell surfacereceptors and/or gene expression markers have been identified. In oneembodiment, the cells express at least one cell surface/gene expressionmarker such as Oct4, nanog, Zfp296, cripto, Gdf3, UtF1, Ecat1, Esg1,Sox2, Pax6, nestin, SCA-1, CD29, CD34, CD90, B1 integrin, cKit, SP-C,CC10, SF1, DAX1, and SCG10. In most embodiments the cells express acombination of markers. Also provided are subpopulations of spore-likecells which can be induced to differentiate into cells of endodermal,mesodermal or ectodermal origin. These subpopulations of spore-likecells, regardless of their tissue of origin, can be used to generatecells originating from all three germ layers.

Methods for identifying and obtaining a subpopulation of spore-likecells are described. The methods include culturing isolated spore-likecells for a period of time sufficient for expression of cell surfaceand/or gene expression markers, and identifying the cell surface and/orgene express markers.

Methods for purifying a subpopulation of spore-like cells of interestfrom a population of spore-like cells are also described. In someembodiments, the methods includes (a) providing a population ofspore-like cells; (b) identifying a subpopulation of the spore-likecells that express one or more markers such as Oct4, nanog, Zfp296,cripto, Gdf3, UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1, CD29, CD34,CD90, B1 integrin, cKit, SP-C, CC10, SF1, DAX1, and/or SCG10; and (c)purifying the sub-population. In some embodiments, the cells areisolated using antibodies specific for the cell surface markers.

Patients who have a disorder, such as a skin disorder, a tumor, or adisease, such as diabetes, can be treated for example, by administeringa subpopulation of spore-like cells to the damaged region (e.g., thedamaged region of the patient's skin, the area from which the tumor wasablated, or the pancreas). Systemic administration is also possible. Themethods can be used to treat a patient who has a deficiency or disorderof functional cells in any of a wide variety of tissues, such as theretina, intestine, bladder, kidney, liver, lung, nervous systemincluding spinal cord or brain, or endocrine system.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows spore-like cell isolated from an insulinoma (tumor frompancreas).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

A pluripotent spore-like cell, as used herein, is a spore-like cell thathas the ability to give rise to cells originating from all three germlayers: mesoderm, endoderm, and ectoderm.

The term “isolated”, as used in the context of a spore-like cell,indicates that the cell exists apart from its native environment. Anisolated cell can be completely isolated from other cell types, or bepresent in an increased amount in a population.

As used herein, an enriched spore-like cell population is a populationof cells which have been isolated from other cells that exist in theirnative environment, so that the proportion of that cell in a mixture ofcells is greater than would be found in its native environment orimmediately following isolation.

As used herein, a “detectable label” refers to any moiety that can beadded to a binding partner of interest, which allows for the detectionof the binding partner.

II. Spore Like Cell Populations

Sources of Spore-Like cells

Spore-like cells can be obtained from an animal donor such as an avianreptilian, amphibian, or mammalian animal. For example, mammalianspore-like cells can be isolated from a rodent, a rabbit, a cow, a pig,a horse, a goat, a sheep, a dog, a cat, a non-human primate, or a human.Spore-like cells can be obtained from a mature animal. Becausespore-like cells tolerate oxygen deprivation better than differentiatedcells, viable spore-like cells can also be isolated from deceasedanimals, including animals that have been deceased for 24 hours or more.Spore-like cells were isolated from chicken livers (avian class)purchased at a grocery store. At initial isolation a trypan blue dyeexclusion test that showed very small viable (dye excluded) spore likecells. Three days following isolation large floating clusters ofspore-like cells too numerous to count, could be seen (data not shown).

Within a given donor, spore-like cells can be obtained from a variety ofsources. For example, spore-like cells can be obtained from bodilyfluids (e.g., blood, saliva, or urine), and most, if not all, functionalorgans. Moreover, spore-like cells can be obtained from the patient whowill be subsequently treated with those cells, from another person, orfrom an animal of a different species. Autologous, allogenic, andxenogeneic subpopulations spore-like cells can be obtained and used totreat patients or to grow tissues.

Spore-like cells can also be isolated from diseased tissue, for example,cancerous tissue (FIG. 1). Spore-like cells thus isolated have asignificant regenerative potential under the right conditions.

Isolation of Spore-Like Cells

Methods for isolating spore-like cells are disclosed in U.S. Pat. Nos.7,060,492, 7,575,921 and 7,560,275 to Vacanti, et al. Briefly, a tissueor blood sample is obtained from an animal. One of the easiest samplesto obtain is a sample of whole blood. Those of ordinary skill in the artwill appreciate that the isolation method may vary slightly depending onthe type of tissue used as the starting material. For example, in theevent the sample is a blood sample, it can be placed in a tubecontaining an anti-coagulant.

After collection, tissue samples, whether they are samples of bodilyfluids or cell suspensions obtained from solid organs, are centrifugedfor a time and at a speed sufficient to pellet the cells within thesample at the bottom of the centrifuge tube. The resulting pellet isre-suspended in a suitable medium such as DMEM/F-12 medium supplementedwith glucose, transferrin, insulin, putricine, selenium, progesterone,epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

The suspended cells are then transferred to a tissue culture vessel andincubated at or near 37° C. Initially, when the sample is a bloodsample, the culture flasks contain primarily hematopoietic cells.However, after several days in culture, the red blood cells lyse so thatthe culture contains primarily, if not exclusively, spore-like cells.This is because the red blood cells have no nucleus; however, thespore-like cells stay alive under these conditions. When spore-likecells are isolated from solid tissues, the differentiated cells can belysed by triturating the sample with a series of pipettes, each having asmaller bore diameter than the one before. For example, the last pipetteused can have a bore diameter of approximately 15μ. After severaladditional days in culture, the spore-like cells multiply and cancoalesce to form clusters (spheres) of cells. Over time, usually on theorder of approximately 7 days, their number can increase greatly.Typically, more than 90% of the cells are viable according to Trypanblue exclusion studies when isolated as described above.

Those of ordinary skill in the art will recognize that triturationthrough reduced bore pipettes is not the only way to isolate spore-likecells from larger, differentiated cells. For example, a suspensioncontaining spore-like cells and differentiated cells can be passedthrough a filter having pores of a particular size. The size of thepores within the filter (and, similarly, the diameter of the pipetteused for trituration) can be varied, depending on how stringent onewishes the isolation procedure to be. Generally, the smaller the poreswithin the filter, or the smaller the diameter of the pipette used fortrituration, the fewer the number of differentiated cells that willsurvive the isolation procedure. Other methods for isolating spore-likecells is freeze/thawing and size exclusion.

In some embodiments a method for isolating spore-like cells fromblood/bone marrow while ensuring exclusion of types of cells/stem cellsinclude: freezing anticoagulated whole blood down to −20 to −80° C.without adding any cyroprotectant agents, then thawing the frozensample. The intracellular ice crystals formed during freezing will killall other cells. The cells are them triturated in culture media and thencultured. The remaining cells that grow are spore-like cells. Othertechniques to isolate spore-like cells from blood include oxygendeprivation for 24 hours. Blood or bone marrow samples are lyses withhypertonic solution to burst cells/stem cells. Not using serum norfeeder cells in your cultures selects for spore-like cells. Thesemethods work well for blood as with other tissues.

Enrichment of Spore-Like Cell Subpopulations

A method for enriching and characterizing, identifying or analyzing theenriched subpopulation of spore-like cells is disclosed. The enrichedsubpopulations are identified using cell surface of gene expressionmarkers expressed by the cells. The cells are cultured for a time periodsufficient for specific marker expression, as demonstrated in theexamples, and marker expression confirmed using techniques known to oneof ordinary skill in the art. Enriched spore-like cell populations arepositive for one or more specific cell markers, such as Oct4, nanog,Zfp296, cripto, Gdf3, UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1,CD29, CD34, CD90, B1 integrin, cKit, SP-C, CC10, SF1, DAX1, and SCG10.The enriched spore-like cell population can be used following detectionof the markers of interest, the cells can be cultured further toencourage additional differentiation, or the cells expressing themarkers can be further isolated from the enriched subpopulation.

Methods for isolating cells expressing a protein of interest for furtherpropagation are known to one of ordinary skill in the art. For example,cells expressing a marker of interest can be isolated using commerciallyavailable ClonePix FL system (Gentix, UK) disclosed in U.S. Pat. No.7,310,147, which works by imaging thousands of cells. Specificfluorescent probes are then used to detect and identify fluorescingcolonies. Cells with the desired level of fluorecence (for example, thehighest producers of a target protein) are then automatically collected(Mann, Nature Methods, i-ii, (2007)). Succesive passage of sphericalclusters of these cells results in a purer population of immature andmore primitive stem cells. In other embodiments, cell surface or geneexpression markers are used to isolate the subpopulations of spore-likecells of interest. In onr embodiment for cell surface markers, themethod includes (a) providing a population of spore-like cells isolatedas described supra; (b) culturing the population of spore-like cells fora period of time sufficient to express the markers; (c) contacting thepopulation of cells with a binding partner that is specific for amarker, and under conditions sufficient to allow binding of the bindingpartner to its target, if present, on each cell of the population ofspore-like cell; and (d) isolating the marker-positive subpopulation.

In some embodiments, in step (c) discussed aboved, the cells arecontacted with a first binding partner that is specific for a firstmarker, and a second binding partner that is specific for a secondmarker which is different from the first marker, under conditionssufficient to allow binding of each binding partner to its target, ifpresent, on each cell of the population of spore-like cells; (d)selecting a first subpopulation of spore-like cells that are positivefor the markers; (e) contacting the first subpopulation of spore-likecells with one or more additional binding partners that are specific forone or more additional cell surface markers under conditions sufficientto allow binding of each binding partner to its target, if present, oneach cell of the population of cells; (f) removing from the firstsubpopulation of cells those cells that bind to at least one of theantibodies of step (e); and (g) collecting a second subpopulation ofcells whereby a subpopulation of spore-like cells is isolated. In someembodiments, the binding partner is an antibody specific for the cellsurface marker.

The isolation of the disclosed subpopulations can be performed using anymethodology that can separate cells based on expression or lack ofexpression of the one or more specific markers including, but notlimited to fluorescence-activated cell sorting (FACS).The markers areselected from the group consisting of Oct4, nanog, Zfp296, cripto, Gdf3,UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1, CD29, CD34, CD90, B1integrin, cKit, SP-C, CC10, SF1, DAX1, SCG10, SCA-1, CD34, CD90, cKIT,B1 integrin.

The separation step can be performed in a stepwise manner as a series ofsteps or concurrently. For example, the presence or absence of eachmarker can be assessed individually, producing two subpopulations ateach step based on whether the individual marker is present. Thereafter,the subpopulation of interest can be selected and further divided basedon the presence or absence of the next marker.

Alternatively, the subpopulation can be generated by separating out onlythose cells that have a particular marker profile, wherein the phrase“marker profile” refers to a summary of the presence or absence of twoor more markers. Each of these individual combinations of markersrepresents a different marker profile. As additional markers are added,the profiles become more complex and correspond to a smaller and smallerpercentage of the original mixed population of cells. In someembodiments, antibodies specific for markers are employed for isolationand/or purification of subpopulations of spore-like cells that havemarker profiles of interest, and it is understood that based on themarker profile of interest, the antibodies can be used to positively ornegatively select fractions of a population, which in some embodimentsare then further fractionated.

In some embodiments, the binding partners are labeled with a detectablelabel. Different binding partners which bind to different markers can belabeled with different detectable labels or can employ the samedetectable label. A variety of detectable labels are known to theskilled artisan, as are methods for conjugating the detectable labels tobiomolecules such as antibodies and fragments and/or derivativesthereof. Representative detectable moieties include, but are not limitedto, covalently attached chromophores, fluorescent moieties, enzymes,antigens, groups with specific reactivity, chemiluminescent moieties,and electrochemically detectable moieties, etc. In some embodiments, thebinding partners are biotinylated. In some embodiments, the biotinylatedbinding partners (the first binding partner) which bind to markers onthe cell surface are detected using a second binding partner comprisingan avidin or streptavidin group that is conjugated to a fluorescentlabel including, but not limited to, Cy3, Cy5, and Cy7. In someembodiments, the binding partner is directly labeled with a fluorescentlabel such as Cy3, Cy5, or Cy7. In some embodiments, the binding partneris directly labeled with a fluorescent label and cells that bind to theantibody are separated by fluorescence-activated cell sorting.Additional detection strategies are known to the skilled artisan.

III. Applications of Subpopulations of Spore-Like Cells

Cell Culture and Genetic Manipulation

The cells disclosed herein can be administered directly to a patient,induced to differentiate into cells of any of endodermal, ectodermal ormesodermal origin prior to administration, or, they can be made toexpress particular factors following genetic manipulation. For example,the subpopulation of spore-like cells can be made to express hormones,such as insulin, by transfecting them with gene constructs that includesequences that encode these factors.

Differentiation of Subpopulations of Spore-Like Cells

The subpopulation of spore-like cells, regardless of their tissue oforigin, can be induced to different into cells from any of the threegerm layers, for example, skin and hair cells including epithelialcells, keratinocytes, melanocytes, adipocytes, cells forming bone,muscle and connective tissue such as myocytes, chondrocytes, osteocytes,alveolar cells, parenchymal cells such as hepatocytes, renal cells,adrenal cells, and islet cells (e.g., alpha cells, delta cells, PPcells, and beta cells), blood cells (e.g., leukocytes, erythrocytes,macrophages, and lymphocytes), retinal cells (and other cells involvedin sensory perception, such as those that form hair cells in the ear ortaste buds on the tongue), nervous tissue including brain and nerves,and fibroblasts.

The subpopulations of spore-like cells can be induced to differentiatein a variety of ways. Marker positive spore-like cells can differentiatewhen they establish contact with a tissue within a patient's body or aresufficiently close to a tissue to be influenced by substances (e.g.,growth factors, enzymes, or hormones) released from the tissue.Differentiation of the cells is influenced by virtue of signals the cellreceives from the surrounding tissue. Such signaling occurs, forexample, when a receptor on the surface of the cell is bound andtransduces a signal from a molecule such as a growth factor, enzyme, orhormone that is released by a tissue within the patient. Alternatively,or in addition, cells disclosed herein can be induced to differentiateby adding a substance (e.g., a growth factor, enzyme, hormone, or othersignaling molecule) to the cell's environment. For example, a substancecan be added to a culture dish containing a subpopulation of spore-likecells, to a mesh or other substrate suitable for applying cells to atissue, or to a tissue within a patient's body. When a substance thatinduces cells to differentiate is administered, either systemically orlocally, it can be administered according to pharmaceutically acceptedmethods. For example, proteins, polypeptides, or oligonucleotides can beadministered in a physiologically compatible buffer, with or without acarrier or excipient. Thus, the subpopulations of spore-like cells candifferentiate either in culture or in a patient's body, and may do sofollowing contact with a solid support or exposure to substances thatare either naturally expressed, exogenously administered, or expressedas a result of genetic manipulation.

In one embodiment, the spore-like cell population is induced todifferentiate into cells of ectodermal origin by exposing the cells toan “ectodermal differentiating” media. In another embodiment thespore-like cells population is induced to differentiate into cells ofmesodermal origin by exposing the cells to “mesodermal differentiatingmedia”. In still another embodiment, the spore-like cell population isinduced to differentiate into cells of endodermal origin by exposing thecells to “endodermal media”. Components of “endodermal”, “mesodermal”and “ectodermal” media are known to one of ordinary skill in the art.Known cell surface markers can be used to verify that the cells areindeed differentiating into cells of the lineage of the correspondingcell culture medium. The most commonly accepted markers to confirmdifferentiation of the three germ layers are the expression of alphafetal protein for endodermal cells, alpha smooth muscle actin formesoderm, and Beta-III tubulin for ectoderm, all of which are normallyexpressed very early in the development of these tissues.

Regardless of the stimulus for differentiation, the subpopulation ofspore-like cells that have differentiated, or that will do so,sufficiently to aid in the maintenance or repair of tissue, can beadministered to a patient (e.g., at the site of a burn or othertraumatized area of skin, a bone fracture, a torn ligament, an atrophiedmuscle, a malfunctioning gland, or an area adversely affected by aneurodegenerative process or autoimmune response).

Tumor Spore-Like Cells

(i) Vaccine Treatment

Subpopulations of spore-like cells obtained may be very useful researchtools and have a potential for therapy, for example, spore-like cellsisolated from a tumor from pancreas may be used with dendritic antigenpresenting cells as a continuous cell mediated vaccine.

Current experimental dendritic vaccines work for a brief period buteventually fail, most likely because they are derived from mature tumorcells. If the tumor cells change genetically, then the vaccine maybecome useless, similar to what occurs with viral influenza vaccinesfrom year to year. Having a powerful evolving tumor stem cell shouldmake for a more effective vaccine. Tumor-derived spore-like cellpopulations may be cultures and used to stimulate cells of the immunesystem to attack and potentially treat tumors or malignancies, asdemonstrated for example in Liau et al., J. of Neuro. 90 (6):1115-24(1999). Liau, et al., (1999) demonstrated that a special type of braintumor (intra cranial glioma) may be treated by actually isolating thesebrain tumor cells and stimulating a special type of immune cell that canbe derived from a patient called a dendritic cell, that actually thenprocesses the antigens produced by the tumor cells and consequentlystimulates the rest of the immune system to attack the actual braintumor resulting in regression or disappearance of the tumor. This attackis mediated through the production of cytokines produced by such immunecells as T-8, T-4 as well as B cells and natural killer cells. Thisparticular approach has shown therapeutic benefit in both animals andearly human trials in treating brain tumors. One of the main limitationsof this technology is that although an effective vaccine can be madeagainst a particular type of tumor cell, over time tumors mutate andconsequently new tumors may arise that will not be responsive to theinitial vaccine. Thus the tumor derived spore-like cells that willexpress a greater variety of antigens and can continuously be kept inculture can be used in stead of tumor cells. As these spore like cellsexpress antigens and mutate, updated vaccines can be synthesized andused to treat tumor recurrences in the patient. We have noted thepresence of spore-like cells in a special type of tumor called aninsulinoma. There is no reason to expect that spore-like cells can notbe isolated from all tumor types. One potential advantage of using tumorderived spore-like cells to stimulate a vaccine is the fact that sincespore-like cells very primitive they have the full potential to expressand differentiate into multiple varieties of tumor cells and expressmultiple antigens. In other words, if a more fundamental tumor cell isused to make a vaccine, then the variety of the immune response to acell that has a larger potential to make different types of tumorantigens, would have an end result in a more powerful vaccine due tonormal mutations that occur in tumors. Additionally spore-like cellsmaybe isolated from the blood, bone marrow and thymus with the capacityto differentiate into all cell types of the immune system includingnatural killer cells, T-4 and T-8 cells, and B or antibody producingcells. Furthermore, dendrocytes (antigen presenting cells) may evolvefrom spore-like cells. This would allow the possible in vitrostimulation of the immune system not only by various types of tumorcells, but also by micro organisms such as viruses, HIV, and bacteriasuch as anthrax. This may provide a mechanism to create new vaccines totumors, and infectious diseases such as HIV and anthrax. Additionallyspore-like cells derived from a tumor by a biopsy maybe cultured invitro and different therapeutic agents can be used to test the effect onthe in vitro malignant spore-like cells thus predicating the patientresponse whether it be a chemotherapeutic agent, vaccine or radiation.Just like bacteria are isolated from sick patients with culture andsensitivities done to choose effective antibiotic treatment, so to willthe isolation of tumor spore like cells from a biopsy sample make tumorculture and chemotherapy sensitivity testing possible. Additionallytumor spore like cells isolated from a patient will be suited for use inthe evolving field of molecular diagnostics and proteonomics, to profilethe patient's tumor for needed diagnosis and therapeutic intervention.

-   -   (ii) DNA Repair in Tumor Cells

DNA in living cells is constantly exposed to damaging agents such asradiation, carcinogens and oxidation. It has been known that damaged DNAis associated with malignant transformation of cells. Consequently cellsare protected from becoming malignant by the presence of DNA repairgenes. For instance, if someone has a genetic predisposition to aparticular type of cancer, in some cases a defect in this DNA repairsystem can be demonstrated. One example is the hereditary non-polyposiscolon cancer syndrome. This consists of a defect in DNA repair that isexpressed as familial cancer of the colon. A nucleotide mismatch canresult in cancer. Normal DNA repair genes would correct this mismatchand consequently tumor formation is averted. Recent stem cell researchhas shown neuronal stem cells to possess the capability of following andtracking down a migrating glioma (a special type of brain tumor) cells.Because of this tumor tracking phenomena, neural stem cells have hadgenes inserted into them for chemicals such as interferon-gamma, tumornecrosis factor and interleukin 12. Upon implantation these unique stemcells were able to track down the brain tumor cells and damage them bysecreting the anti-tumor chemical. Additionally recent literature hasdemonstrated that adult stem cells have the capacity to fuse with normalcells when placed in the body. Disclosure relevant to DNA repair asdiscussed above can be found for example in Ourednik, et al., NatureBiotechnol., 20 (11): 1103-1110 (2002); Stewart, et al., Bioessays, 24(8): 708-13 (2002); Ehtesham, et al., Cancer Therapy, 9 (11): 925-934(2002); Ehtesham, et al., Cancer Reserves, 62 (20): 5657-63 (2002);Ferguson, et al., Oncogene 20(40): 5572-9 (2001).

The spore-like cells populations disclosed herein can therefore be usedto treat tumors using the unique properties of seeking out malignantcells by tracking, fusion, and DNA repair. In vitro spore-like cellshave been observed to cluster and fuse with normal cells that are grownin culture with the spore-like cell. Spore-like cells can easily beisolated from non-prepared anticoagulated frozen blood and expanded inculture. As opposed to neural stem cells these spore-like cells areeasily derived, especially from the blood and rapidly increase innumbers when incubated. Additionally, they are small consequently havinga greater opportunity to fuse with tumor cells because of their greatnumbers, small size and very simple structure. Previous descriptionsshow essentially a very small simple structure or packets of membranebound DNA. All cells have natural DNA repair systems. An example wouldbe endonucleases that can cut out damaged, defective DNA and thenreplace these damaged DNA segments with normal DNA using DNApolymerases. As cells age, their DNA repair systems fail with consequentmalignant transformation. Most therapeutic modalities for cancer focuson surgical removal of the tumor and the use of tumor cell killingmechanisms such as chemotherapy, radiation, or immunotherapy. Theseapproaches are associated with significant morbidity and are not alwayseffective. There are no approaches of using DNA repair to convert atumor cell into a normal cell, even though these repair systems areknown to exist, and their failure is involved in the causing of cancer.The infusion of billions of spore like cells derived from the patient'sown blood, with fresh DNA repair systems that can seek out and trackdown tumor cells would have the capacity to fuse with the tumor cellsand donate their DNA repair systems to the defective tumor cells. Theymay also exert a repair effect on the tumor cells by secreting agentswith trophic effects. These spore-like cells could be directly infusedinto the tumor or given by IV or IP routes. Since this would be anaugmentation of natural DNA repair systems known to exist, side effectswould be minimal compared to more traditional cell suicidal approachessuch as chemotherapy. Essentially an unlimited number of spore-likecells could be generated from the patient's own blood to accomplish thistask.

Additionally, gene insertion technology can be used to insert nucleotidesequences into spore like cells that express anti-tumor substances, suchas interferon gamma and tumor necrosis factor. Once infused, thesegenetically engineered spore-like cells would track down and fuse withthe tumor cells delivering the interferon, or TNF directly to the tumorwith therapeutic effect. This represents an improvement in prior art,because spore-like cell populations can be expanded in vitro to supplyan inexhaustible amount of simple cell structures that can track down,fuse and consequently repair the damaged DNA found in tumor cells. Priorart has demonstrated that neural stem cells possess these properties,but harvesting neural stem cells from a patient maybe associated withmany risks. This treatment approach may have uses for cancers of thebreast, lung, brain, and prostrate as well as other organs.

(iii) Reversal of Degenerative Diseases

Gene based therapeutics or gene insertion technology involves effectivedelivery of specific nucleotide sequences or genes will result in aclinical improvement because of the production of a needed proteinproduced by the specific gene. Potential uses include treatmentmodalities for cancer, vaccines, genetic and metabolic disorders, andother areas including viral infections, cardiovascular disease and otherorgan disease states. One of the major limitations of gene basedtherapeutics has been finding an adequate and effective delivery system.Much work has been done in regards to gene insertion and adenoviralvectors with the consequent infection of the patient resulting in theexpression of the needed protein because of insertion of the neededtherapeutic gene. Problems have included ineffective gene insertion intothe desired host cells as well as hazardous effects of the viral vectoractually infecting the host. As demonstrated in the example, spore-likecell populations can be easily isolated from the patient including bloodexpanded in vitro; the spore-like cells can be safely returned to thepatient by various routes such as IV, intraperitoneal or subcutaneouslyto name a few. Genes encoding proteins such as Factor 8 (for treatmentof hemophilia) can be inserted in an in vitro environment with expansionof the spore-like cell population with the new therapeutic gene andproduct using techniques such as those disclosed in Vacanti, et al.,Transplantation Proceedings, 33:592-598 (2001). Additional evidence ofpotential use includes the expression of Brdu, indicating active nucleicacids synthesis. This method may also allow the actual in vitroproduction of needed therapeutic proteins such as Factor 8 utilizing thespore-like cell population in recombinant techniques with the consequentproduction of a therapeutic protein. Ideally, a permanent in vivo effectmay be possible. For examples, blood spore like cell populations can beisolated from a patient with hemophilia or Tay Sach's disease, a geneinserted in vitro, then the corrected cells could be safely implantedback into the patient with proliferating cells with the potential tocorrect the enzyme deficiency and cure the disease. Any known geneenzyme deficiency resulting in a disease may be amenable to treatmentusing this approach. Potentially treatable genetic diseases includeHuntington's Disease, muscular dystrophy, familial hypercholesterolemia,cystic fibrosis, phenylketonuria, hemachromatosis, sickle cell anemia,and glycogen storage disease to name a few. With the mapping of thehuman genome, eventually most the genetic mutations resulting in diseasewill be worked out opening up the opportunity for treatment withappropriate gene insertion technology. Autologous derived spore likecell populations may provide the needed vehicle of delivery of thesecorrected genes. The properties of their small size and exuberantability to divide and to allow the insertion of therapeutic nucleicacids sequences with expression, indicates that this is a reasonableapplication. The gene insertion may occur into the spore-like cell invitro with just naked DNA alone which includes the needed gene sequence.This would avert any sort of potential hazards involving the use ofviral vectors such as the adenoviral technique. Using this mode of geneinsertion may provide additional modalities of therapy such as insertingnucleic acid sequences to make therapeutic agents such as a tumorsuppressor protein, or an angiogenesis inhibitor to stop tumors fromgrowing. Another gene which could be inserted into the spore-like cellpopulations includes a vasoactive gene such as VEGF to treat ischemicheart disease thus avoiding the need for bypass heart surgery. The CFRTgene which will make a protein to treat cystic fibrosis may be expressedby spore-like cells that have traveled to the lung through thecirculation or have been injected into the lung through the bronchi withconsequent engraftment, and differentiation in the lung tissue withgeneration of a needed gene product to help treat the symptoms of cysticfibrosis.

If bulk production of a needed product is desired the spore like cellcould be implanted with biodegradable scaffolding material, hydrogels,or a naturally derived matrix such as we have isolated from frozen bloodand other tissues such as heart, lung and CSF. A prior limitation ofgene insertion therapy has been over time the amount of product dwindlesprobably due to dissipation of corrected cells into the body andeventual disappearance. The production of a tissue construct using thespore-like cell population with inserted gene nucleotides on a naturalor bio-degradable scaffolding would be a way to overcome thislimitation.

Structures and Formulations for Administration of Cells

The cell populations can be administered to a patient by way of acomposition that includes a population of spore-like cells alone or onor in a carrier or support structure. In many embodiments, no carrierwill be required. The spore-like cells will be administered by injectiononto or into the site where the cells are required. In these cases, thecells will typically have been washed to remove cell culture media andwill be suspended in a physiological buffer.

In other embodiments, the cells will be provided with or incorporatedonto or into a support structure. Support structures may be meshes,solid supports, tubes, porous structures, and/or a hydrogel. The supportstructures may be biodegradable or non-biodegradable, in whole or inpart. The support may be formed of a natural or synthetic polymer, metalsuch as titanium, bone or hydroxyapatite, or a ceramic. Natural polymersinclude collagen, hyaluronic acid, polysaccharides, andglycosaminoglycans. Synthetic polymers include polyhydroxyacids such aspolylactic acid, polyglycolic acid, and copolymers thereof,polyhydroxyalkanoates such as polyhydroxybutyrate, polyorthoesters,polyanhydrides, polyurethanes, polycarbonates, and polyesters.

Solid Supports

The support structure may be a loose woven or non-woven mesh, where thecells are seeded in and onto the mesh. The structure may include solidstructural supports. The support may be a tube, for example, a neuraltube for regrowth of neural axons. The support may be a stent or valve.The support may be a joint prosthetic such as a knee or hip, or partthereof, that has a porous interface allowing ingrowth of cells and/orseeding of cells into the porous structure.

The support structure may be a permeable structure having pore-likecavities or interstices that shape and support the hydrogel-cellmixture. For example, the support structure can be a porous polymermesh, a natural or synthetic sponge, or a support structure formed ofmetal or a material such as bone or hydroxyapatite. The porosity of thesupport structure should be such that nutrients can diffuse into thestructure, thereby effectively reaching the cells inside, and wasteproducts produced by the cells can diffuse out of the structure

The support structure can be shaped to conform to the space in which newtissue is desired. For example, the support structure can be shaped toconform to the shape of an area of the skin that has been burned or theportion of cartilage or bone that has been lost. Depending on thematerial from which it is made, the support structure can be shaped bycutting, molding, casting, or any other method that produces a desiredshape. The support can be shaped either before or after the supportstructure is seeded with cells or is filled with a hydrogel-cellmixture, as described below.

Additional factors, such as growth factors, other factors that inducedifferentiation or dedifferentiation, secretion products,immunomodulators, anti-inflammatory agents, regression factors,biologically active compounds that promote innervation or enhance thelymphatic network, and drugs, can be incorporated into the polymersupport structure.

An example of a suitable polymer is polyglactin, which is a 90:10copolymer of glycolide and lactide, and is manufactured as VICRYL™braided absorbable suture (Ethicon Co., Somerville, N.J.). Polymerfibers (such as VICRYL198 ), can be woven or compressed into a felt-likepolymer sheet, which can then be cut into any desired shape.Alternatively, the polymer fibers can be compressed together in a moldthat casts them into the shape desired for the support structure. Insome cases, additional polymer can be added to the polymer fibers asthey are molded to revise or impart additional structure to the fibermesh. For example, a polylactic acid solution can be added to this sheetof polyglycolic fiber mesh, and the combination can be molded togetherto form a porous support structure. The polylactic acid binds thecrosslinks of the polyglycolic acid fibers, thereby coating theseindividual fibers and fixing the shape of the molded fibers. Thepolylactic acid also fills in the spaces between the fibers. Thus,porosity can be varied according to the amount of polylactic acidintroduced into the support. The pressure required to mold the fibermesh into a desirable shape can be quite moderate. All that is requiredis that the fibers are held in place long enough for the binding andcoating action of polylactic acid to take effect.

Alternatively, or in addition, the support structure can include othertypes of polymer fibers or polymer structures produced by techniquesknown in the art. For example, thin polymer films can be obtained byevaporating solvent from a polymer solution. These films can be castinto a desired shaped if the polymer solution is evaporated from a moldhaving the relief pattern of the desired shape. Polymer gels can also bemolded into thin, permeable polymer structures using compression moldingtechniques known in the art.

Many other types of support structures are also possible. For example,the support structure can be formed from sponges, foams, corals, orbiocompatible inorganic structures having internal pores, or mesh sheetsof interwoven polymer fibers. These support structures can be preparedusing known methods.

Hydrogels

In another embodiment, the cells are mixed with a hydrogel to form acell-hydrogel mixture. This cell-hydrogel mixture can be applieddirectly to a tissue that has been damaged. For example, as described inU.S. Pat. No. 5,944,754 a hydrogel-cell mixture can simply be brushed,dripped, or sprayed onto a desired surface or poured or otherwise madeto fill a desired cavity or device. The hydrogel provides a thin matrixor scaffold within which the cells adhere and grow. These methods ofadministration may be especially well suited when the tissue associatedwith a patient's disorder has an irregular shape or when the cells areapplied at a distant site (e.g., when spore-like cells are placedbeneath the renal capsule to treat diabetes).

Alternatively, the hydrogel-cell mixture can be introduced into apermeable, biocompatible support structure so that the mixtureessentially fills the support structure and, as it solidifies, assumesthe support structure's shape. Thus, the support structure can guide thedevelopment and shape of the tissue that matures from spore-like cells,or their progeny, that are placed within it. As described further below,the support structure can be provided to a patient either before orafter being filled with the hydrogel-cell mixture. For example, thesupport structure can be placed within a tissue (e.g., a damaged area ofthe skin, the liver, or the skeletal system) and subsequently filledwith the hydrogel-cell composition using a syringe, catheter, or othersuitable device. When desirable, the shape of the support structure canbe made to conform to the shape of the damaged tissue. In the followingsubsections, suitable support structures, hydrogels, and deliverymethods are described.

The hydrogels should be biocompatible, biodegradable, capable ofsustaining living cells, and, preferably, capable of solidifying rapidlyin vivo (e.g., in about five minutes after being delivered to thesupport structure). Large numbers of cells can be distributed evenlywithin a hydrogel. A hydrogel can normally support approximately 5×10⁶cells/ml. Hydrogels also enable diffusion so that nutrients reach thecells and waste products can be carried away.

A variety of different hydrogels can be used. These include, but are notlimited to: (1) temperature dependent hydrogels that solidify or set atbody temperature (e.g., PLURONICS™.); (2) hydrogels cross-linked by ions(e.g., sodium alginate); (3) hydrogels set by exposure to either visibleor ultraviolet light, (e.g., polyethylene glycol polylactic acidcopolymers with acrylate end groups); and (4) hydrogels that are set orsolidified upon a change in pH (e.g., TETRONICS™). Methods for thesynthesis of the other polymers described above are known to those ofordinary skill in the art. See, for example Concise Encyclopedia ofPolymer Science and Engineering, J. I. Kroschwitz, Ed., John Wiley andSons, New York, N.Y., 1990. Many polymers, such as poly(acrylic acid),alginates, and PLURONICS™ are commercially available.

Ionic polysaccharides, such as alginates or chitosan, can be used tosuspend living cells, including spore-like cells and their progeny.These hydrogels can be produced by cross-linking the anionic salt ofalginic acid, a carbohydrate polymer isolated from seaweed, with ions,such as calcium cations. The strength of the hydrogel increases witheither increasing concentrations of calcium ions or alginate. U.S. Pat.No. 4,352,883 describes the ionic cross-linking of alginate withdivalent cations, in water, at room temperature, to form a hydrogelmatrix.

The subpopulations of spore-like cells are mixed with an alginatesolution. The solution is delivered to an already implanted supportstructure, which then solidifies in a short time due to the presence ofphysiological concentrations of calcium ions in vivo. Alternatively, thesolution is delivered to the support structure prior to implantation andsolidified in an external solution containing calcium ions.

In general, these polymers are at least partially soluble in aqueoussolutions (e.g., water, aqueous alcohol solutions that have charged sidegroups or monovalent ionic salts thereof). There are many examples ofpolymers with acidic side groups that can be reacted with cations (e.g.,poly(phosphazenes), poly(acrylic acids), and poly(methacrylic acids)).Examples of acidic groups include carboxylic acid groups, sulfonic acidgroups, and halogenated (preferably fluorinated) alcohol groups.Examples of polymers with basic side groups that can react with anionsare poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl imidazole).

Water soluble polymers with charged side groups are cross-linked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups, or multivalent anions if the polymer has basicside groups. Cations for cross-linking the polymers with acidic sidegroups to form a hydrogel include divalent and trivalent cations such ascopper, calcium, aluminum, magnesium, and strontium. Aqueous solutionsof the salts of these cations are added to the polymers to form soft,highly swollen hydrogels.

Anions for cross-linking the polymers to form a hydrogel includedivalent and trivalent anions such as low molecular weight dicarboxylateions, terepthalate ions, sulfate ions, and carbonate ions. Aqueoussolutions of the salts of these anions are added to the polymers to formsoft, highly swollen hydrogels, as described with respect to cations.

For purposes of preventing the passage of antibodies into the hydrogel,but allowing the entry of nutrients, a useful polymer size in thehydrogel is in the range of between 10 and 18.5 kDa. Smaller polymersresult in gels of higher density with smaller pores.

Temperature-dependent, or thermosensitive, hydrogels can also be used.These hydrogels have so-called “reverse gelation” properties, i.e., theyare liquids at or below room temperature, and gel when warmed to highertemperatures (e.g., body temperature). Thus, these hydrogels can beeasily applied at or below room temperature as a liquid andautomatically form a semi-solid gel when warmed to body temperature. Asa result, these gels are especially useful when the support structure isfirst implanted into a patient, and then filled with the hydrogel-cellcomposition. Examples of such temperature-dependent hydrogels arePLURONICS™ (BASF-Wyandotte), such as polyoxyethylene-polyoxypropyleneF-108, F-68, and F-127, poly (N-isopropylacrylamide), andN-isopropylacrylamide copolymers.

These copolymers can be manipulated by standard techniques to affecttheir physical properties such as porosity, rate of degradation,transition temperature, and degree of rigidity. For example, theaddition of low molecular weight saccharides in the presence and absenceof salts affects the lower critical solution temperature (LCST) oftypical thermosensitive polymers. In addition, when these gels areprepared at concentrations ranging between 5 and 25% (W/V) by dispersionat 4° C., the viscosity and the gel-sol transition temperature areaffected, the gel-sol transition temperature being inversely related tothe concentration. These gels have diffusion characteristics capable ofallowing spore-like cells and their progeny to survive and be nourished.

U.S. Pat. No. 4,188,373 describes using PLURONIC™ polyols in aqueouscompositions to provide thermal gelling aqueous systems. U.S. Pat. Nos.4,474,751, 4,474,752, 4,474,753, and 4,478,822 describe drug deliverysystems that utilize thermosetting polyoxyalkylene gels. With thesesystems, both the gel transition temperature and/or the rigidity of thegel can be modified by adjustment of the pH and/or the ionic strength,as well as by the concentration of the polymer.

Other suitable hydrogels are pH-dependent. These hydrogels are liquidsat, below, or above specific pH values, and gel when exposed to specificpHs, for example, 7.35 to 7.45, the normal pH range of extracellularfluids within the human body. Thus, these hydrogels can be easilydelivered to an implanted support structure as a liquid andautomatically form a semi-solid gel when exposed to body pH. Examples ofsuch pH-dependent hydrogels are TETRONICS™ (BASF-Wyandotte)polyoxyethylene-polyoxypropylene polymers of ethylene diamine,poly(diethyl aminoethyl methacrylate-g-ethylene glycol), andpoly(2-hydroxymethyl methacrylate). These copolymers can be manipulatedby standard techniques to affect their physical properties.

Other hydrogels that can be used to administer spore-like cells or theirprogeny are solidified by either visible or ultraviolet light. Thesehydrogels are made of macromers including a water soluble region, abiodegradable region, and at least two polymerizable regions (see, e.g.,U.S. Pat. No. 5,410,016). For example, the hydrogel can begin with abiodegradable, polymerizable macromer including a core, an extension oneach end of the core, and an end cap on each extension. The core is ahydrophilic polymer, the extensions are biodegradable polymers, and theend caps are oligomers capable of cross-linking the macromers uponexposure to visible or ultraviolet light, for example, long wavelengthultraviolet light.

Examples of such light solidified hydrogels include polyethylene oxideblock copolymers, polyethylene glycol polylactic acid copolymers withacrylate end groups, and 10K polyethylene glycol-glycolide copolymercapped by an acrylate at both ends. As with the PLURONIC™ hydrogels, thecopolymers comprising these hydrogels can be manipulated by standardtechniques to modify their physical properties such as rate ofdegradation, differences in crystallinity, and degree of rigidity.

Hydrogels may be administered by injection or catheter, or at the timeof implantation of other support structures. Crosslinking may occurprior to, during, or after administration.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

EXAMPLES

Using the methods described in the patent application, populations ofspore like cells were isolated from bone marrow as well as severaltissues representing endoderm, mesoderm and ectoderm. Immediately afterisolation, the cells were placed in a media referred to as “basic media”composed of DMEM/F12 with antibiotics B27, FGF and EGF. The cells weremaintained in the basic media for several weeks during which timesequential studies were performed to identify markers expressed by thesecells. Markers were identified using both gene expression analysis andimmunohistochemistry to identification of protein expression.

Spore-like cells acquired from tissues representative of endoderm,mesoderm and ectoderm were analyzed for expression of markers associatedwith embryonic stem cells, while exposed to the “basic media”.Populations of spore-like cells procured from endodermal tissue (lungand liver), mesodermal tissue (muscle) and ectodermal tissue (spinalcord and adrenal gland), all expressed the same panel of genetic andprotein markers as the bone marrow acquired spore-like cells, as well asabsence of the same markers as bone marrow derived spore-like cellswhile maintained in the “basic media”.

Cells were then exposed to one of three differentiating media: Endodermdifferentatiating media, Mesoderm differentatiating media, and Ectodermdifferentatiating media. The composition of the media is as follows:

Endoderm differentatiating media: Hepatocyte culture media ascorbicacid, BSA-FAF, hydrocortisone, transferrin, insulin, hEGF, genomyosin,plus 10% fetal bovine serum.

Mesoderm differentatiating media: DMEM plus 20% fetal bovine serum

Ectoderm differentatiating media: 10% fetal bovine serum added to thebasic media composed of DMEM/F12 plus B27, BEDA FGF, EGF.

When the spore-like cell populations, including disaggregated clustersacquired from bone marrow and from each of the representative tissues,were exposed to each of the “differentatiating medias” supplemented asdescribed above, the cells differentiated into cells representative ofeither endoderm, mesoderm or ectoderm, as a function of the media used,as determined by the expression of known protein and genetic markers,i.e., when an Endoderm differentiating media was used, the cellsexpressed the following protein markers consistent with endodermaltissue: cytokeratin 18, albumin and alpha fetal protein. The geneticmarker, GATA4 was also expressed.

When a Mesoderm differentiating media was used, the cells expressed thefollowing protein markers consistent with Mesodermal tissue: Myosin,desmin, alpha smooth muscle actin. The genetic marker burachyury wasalso expressed.

When an Ectoderm differentatiating media was used, the cells expressedthe following protein markers consistent with Ectodermal tissue: Nestin,Map2 Beta-III tubulin (both of which are markers of neurons), 04 (amarker of oligodendrocytes), GFAP s (a marker of glial cells).Expression of Map2 was also verified by gene expression analysis.

As would be understood by one of ordinary skill in the art, the mostcommonly accepted markers to confirm differentiation of the three germlayers are the expression of alpha fetal protein for endoderm, alphasmooth muscle actin for mesoderm, and Beta-III tubulin for ectoderm, allof which are normally expressed very early in the development of thesetissues. In addition, the genetic markers map2, burachyury and gata4 areexpressed quite early in the germ layer formation of the embryo.

Results

(i) Bone Marrow Derived Spore-Like Cells:

Spore-like cell populations isolated from the bone marrow generatedmulti cellular clusters within several days. Individual cells, eithercontained within the clusters, or alone expressing the following geneticmarkers, all of which have been associated with embryonic stem cells:Oct4, Nanog, Dax1, Fgf4, Zfp296, Cripto, Gdf3, Utf1, Cdx2, Ecat1, Esg1,Sox2 and Pax6, cMyc (a proliferative gene), Fgf5, Olig2 and Pdgfr12.Also the marker nestin (a marker generally associated with neural stemcells) was noted.

Oct4 is a POU transcription factor and nanog is an NK-2 class homeboxtranscription factor, both of which have been associated with themaintenance of the undifferentiated state and pluripotency in embryonicstem cells. (Pan, et al., Cell Res., 12:321-329 (2002); Clark, et al.,Stem Cells, 22: 169-179 (2004); Niwa, Development, 134:635-646 (2007)).Cripto (teratocarcinoma-derived growth factor 1), a marker expressedearly in the inner cell mass, is a multifunctional cell surface proteinwith important roles in vertebrate embryogenesis (Strizzi, Oncogene,24:5731-5741 (2005)). GDF3 is a member of the TGFβ super family ofligands that is specifically expressed in the pluripotent state (Levine,Development, 133:209-16 (2005)). Undifferentiated embryonictranscription factor (UtF1) was first identified as a transcriptionco-factor that is expressed in mouse embryonic stem cells in a stem-cellspecific manner. Utf1 is presumed marker for pluripotency (Niwa, CellStructure and Function, 26(3):137-148 (2001)). Embryonic stem cellassociated transcript 1 (Ecat1) is an embryonic stem cell-associatedtrans gene. Embryonic stem cell-specific gene (Esg1) which encodes aKH-domain containing protein, is expressed in pluripotent cells,including ES cells, embryonic germ cells, and multipotent germ line stemcells (Kanatsu-Shinohara, Cell, 1001-1012 (2004)). SRY (sex determiningregion Y)-box 2, also known as Sox2 and Pax6 are markers associated withboth ES cells and neural stem cells. Sox2 is one of the keytranscription factors required in induced pluripotent stem cells (Zhao,J. Cell. Biochem. 105(4): 949-55 (2008)). Nestin is a class VIintermediate filament protein expressed predominantly in stem cells fromthe central nervous system (Frederisken, et al., J. Neurosci., 8:1144-51(1988)). Thus, the spore-like cells obtained from the bone marrowexpress markers indicative of pluripotency.

The cells also expressed the following protein markers associated withembryonic stem cells: C-kit and Sca1 (surface markers) and E-cadherin asdemonstrated by immunohistochemistry.

Of note is the fact that the spore-like cells isolated from the bonemarrow stained negative for the following embryonic stem cell markers:Dax1, fibroblast growth factor 4 (Fgf4), caudal type homeoboxtranscription factor 2 (Cdx2), Neo and Eras (a tumor formation gene ofES), and Rex 1. The cells also stained negative for P75 a neural creststem cell marker. Nor did they express the protein markers: Cd45 Cd31 orCd34 (endothelial and hematopoietic stem cell markers).

(ii) Muscle

Spore-like cells obtained from muscle initially expressed the sameembryonic stem cell markers observed with the spore-like cells obtainedfrom the bone marrow, i.e., the cells expressed Oct4, Dax1, Fgf4,Zfp296, Cripto Gdf3, Utf1, Cdx2, Ecat1, Esg1, Sox2 and Pax6, cMyc, Fgf5,Olig2, Pdgfr12.

The muscle-derived spore-like cells, like the bone-marrow derivedspore-like cells, stained negative for Dax1, Fgf4, Cdx2, Neo and Eras,Rex 1 (embryonic stem cell markers), P75 (neural crest marker, Cd45 orCd31 (endothelial and hematopoietic stem cell markers).

The spore-like cells isolated from muscle were studied after they formedmulti cellular clusters (in the form of spheres).

The spheres started to form as early as a few days, but were not testedfor markers for at least 3 weeks after initial isolation. The spheresultimately grew to sizes of more than 300 microns in diameter. Spheresformed from muscle spore-like cells stained positive for the followingmarkers: ckit, a cell surface marker indicating a stem cell, notnecessarily an embryonic stem cell (ESC)). Almost 50% of the cells inthe spheres were positive at 3 weeks, however, at 90 days, only 1-5%were positive (this indicates that they were losing their “stemcellness”, as they matured).

Cells shed from myospheres were also analyzed. These are cells that arederived and shed from the myospheres as they grow and mature. Antibodiesfor Stem cell antigen 1 (Sca-1), CD31, and CD45 were all directlyconjugated to PE. Antibodies for CD29, CD34, and CD90 were all directlyconjugated to FITC. Cells in the spheres were positive for the followingmarkers: SCA-1, CD29, CD34, CD90 and B1 integrin, while they werenegative for CD31, CD45, which are hematopoietic and endothelial cellmarkers of more mature cells.

Marker expression changed with the passage of time. As cells that wereshed from the spheres attached, they lost the immature marker CD90first, then started to lose Sca-1. Also, myosphere cells did not expressthe mature cell marker, MyoD and expressed very little or no Pax7 andMyf5.

However when these myospheres were plated, adherent cells matured toexpressed Pax7, MyoD, and Desmin and after adding in differentiationmedia fused to form multinucleated myotubes. Myosphere cells plated inadipogenic and osteogenic media differentiated into fat and bone. Whenmyosphere derived cells were labeled with GFP and then injected intomuscle, they generated GFP labeled muscle fibers. Cells generatedthrough the formation of myospheres are multipotent stem cells sincethey do not yet express myogenic markers such as: Pax7, Myf5, and MyoD,yet they express these markers as they mature, and are able to formmulti-nucleated myotubes, adipogenic and osteogenic cells, and canregenerate injured muscle in vivo. The staining for MyoD (nuclear),Desmin (cytoplasmic) and Sca 1 (surface) was shown in yellow-red. Thestaining for Pax7 (nuclear) was shown in green. The findings wereconfirmed with reverse transcriptase PCR in that myospheres arepre-myogenic, and they do not express Pax7, Myf5, or MyoD. While cellsthat shed from the myospheres attached and then matured to express Pax7,Myf5, and MyoD.

(iii) Lung

A similar time-dependent expression of markers was observed withspore-like cells isolated from the lung. Spore-like cells isolated fromthe lung initially express cKit, and then Nestin.

Later, cells shed from the spheres expressed SP-C, a marker for type IIepithelial cells in alveoli (gene expression and protein analysis), andCC10 for Clara cells (gene expression and protein analysis).

(iv) Adrenal Gland

Adrenal gland expresses very early stem cell markers then matures toexpress markers of chromaffin cells and cell that make catecholamines.When initially isolated, spore-like cells obtained from the adrenalgland, like the cells from the bone marrow, expressed Oct4, Dax1, Fgf4,Zfp296, Cripto Gdf3, Utf1, Cdx2, Ecat1, Esg1, Sox2 and Pax6, cMyc, Fgf5,Olig2, Pdgfr12.

The adrenal gland-derived spore-like cells similarly stained negativefor Dax1, Fgf4, Cdx2, Neo and Eras, Rex 1 (embryonic stem cell markers),P75 (neural crest marker, Cd45 or Cd31 (endothelial and hematopoieticstem cell markers).

In time the mature cell markers were expressed: SF1, a steroidogeneticmarker of the adrenal cortex, DAX1, SCG10 progenitor, cromaffin.

(v) Brain and Peripheral Nervous System

Brain and peripheral nervous system include: brain, spinal cord andperipheral nerve. In these tissues we have identified embryonic markersSca-1, and Oct-4. They mature to show nestin, other neuro markers and amarker for Schwann cells in peripheral nerve.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. An isolated sub-population of spore-like cells.
 2. Thesub-population of spore-like cells of claim 1 expressing one or moremarkers selected from the group consisting of Oct4, nanog, Zfp296,cripto, Gdf3, UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1, CD29, CD34,CD90, B1 integrin, cKit, SP-C, CC10, SF1, DAX1, and SCG10.
 3. Thesub-population of spore-like cells of claim 2, wherein the cells areisolated from post-natal animal tissue selected from the groupconsisting of ectodermal, mesodermal and endodermal tissue.
 4. Thesubpopulaton of spore-like cells of claim 3, wherein the post-natalanimal tissue is selected from the group consisting of mammalian, avian,reptilian and amphibian tissue.
 5. The subpopulation of spore-like cellsof claim 4, wherein the tissue is selected from the group consisting ofheart, intestine, bladder, kidney, liver, lung, adrenal gland, skin,retina, and pancreas.
 6. A method of isolating a subpopulation ofspore-like cells comprising the steps of: (a) isolating spore-like cellsfrom a tissue (b) culturing the cells in for a time sufficient forexpression of one or more markers selected from the group Oct4, nanog,Zfp296, cripto, Gdf3, UtF1, Ecat1, Esg1, Sox2, Pax6, nestin, SCA-1,CD29, CD34, CD90, B1 integrin, cKit, SP-C, CC10, SF1, DAX1, and SCG10,and (c) identifying and isolating cells expressing the one or moremarkers.
 7. The method of claim 6 wherein the cells are isolated usingantibodies specific for the marker.
 8. The method of claim 7 comprisingthe steps of: (a) contacting the population of cells with a bindingpartner that is specific for a marker, and under conditions sufficientto allow binding of the binding partner to its target, if present, oneach cell of the population of spore-like cell; and (b) isolating themarker-positive subpopulation.
 9. The method of claim 8 wherein thecells are isolated using fluorescence-activated cell sorting.